The present invention relates to a biologically active serum-derived composition of matter (SDF), having a low molecular weight, being electrically charged at acidic pH and having absorption at 280 nm, to methods for the isolation thereof and to pharmaceutical compositions comprising the same.
Normal hemopoiesis is coordinated by a variety of regulators which include glycoprotein growth factors (cytokines), such as the colony stimulating factors, as well as non-protein small molecules such as the retinoids. They regulate the survival (apoptosis), proliferation and differentiation of progenitor and precursor cells and the activation state of mature cells. Both proliferation and differentiation processes are regulated by positive and negative stimuli. In acute leukemia, a block in the cell differentiation leads to a massive accumulation of proliferative, undifferentiated non-functional cells. Recently, these regulators have been used in a wide array of clinical and laboratory applications. For example, cytokines are used for treatment of patients with aplastic states such as post bone marrow (BM) transplantation, radio-chemotherapy etc., as well as for ex vivo expansion of specific subsets of cells valuable in cell therapy (transplantation, immuno- or gene-therapy). Low molecular weight compounds, such as retinoids, have been used for induction of differentiation in leukemic cells as a therapeutic modality.
The current approach to treatment of leukemia is based on killing the malignant cells by chemo- or radiotherapy. Such treatment is not specific to the malignant cells and damages also dividing normal cells. Therefore, an alternative approach is being developed, based on inducing the undifferentiated leukemic cells to undergo differentiation. Evidently, terminal differentiation of hemopoietic cells is associated with loss of leukemogenicity.
It has been shown that some undifferentiated myeloid leukemia cells respond to cytokines (e.g. IL-6) and undergo differentiation into mature, functional, non-dividing granulocytes or macrophages, and thereby lose their leukemogenic potential [Fibach, E., et al., Nature, New Biology 237:276 (1972); Shabo, Y., et al., Blood 78:2070 (1988); Fibach, E., et al., Proc. Natl. Acad. Sci. USA 70:343-346 (1973); Inbar, M., et al., Proc. Natl. Acad. Sci. USA 70:2577-2581 (1973); Fibach, E. and Sachs, L. J., Cell Physiol. 83:177-185 (1974); Hayashi, M., et al., Int. J. Cancer 14: 40-48 (1974); Fibach, E. and Sachs, L. J., Cell Physiol. 86:221-230 (1975); Fibach. E. and Sachs, L. J., Cell Physiol. 89:259-266 (1975)].
Other differentiation inducers include dimethylsulfoxide, hexamethylene bis-acetamide, butyric acid [Collins, S. J., et al., Proc. Natl. Acad. Sci. USA 75:2458 (1978)], derivatives of vitamins A and D3 [Breitman, T. T., et al., Proc. Natl. Acad. Sci. USA 77:2936 (1980)] and low doses of cytotoxic drugs such as actinomycin D and cytosine arabinoside [Breitman, T. T., et al., ibid.]. Retinoic acid has been used in the treatment of acute promyelocytic leukemia [Chomienne, C., FASEB 10: 1025 (1996)].
Although capable of inducing some cell lines, these inducers have only rarely been found to induce terminal differentiation in cells freshly isolated from leukemic patients [Breitman et al., (1980) ibid.].
Several publications describe some activities of the high molecular weight, copper binding protein, ceruloplasmin (CP) in malignant and aplastic states.
For example, in JP 56120622 and JP 56090015, CP is described as the active ingredient in an antitumor preparation against leukemia. JP 56120622 describes the CP as having a therapeutic activity against several mammalian tumors due to its inhibitory effect on aggravation of cancer. In addition, CP is described as being capable of inactivating the strong oxidative super-oxide anion radicals by converting them into oxygen molecules. It is also mentioned that CP has an effect on the promotion of liver catalase biosynthesis.
JP56090015 describes a preventative and remedial drug for side reactions of anti-malignant tumor agents, which contains human CP as the main ingredient.
JP 56002916 also describes CP as an anti-tumor agent. This publication is concerned with compositions for the prevention and treatment of radiation damages which contain CP as the active ingredient. Animals irradiated with xcex3-rays, after pre-incubation with a composition comprising CP, showed a high survival rate. The preventive activity described in this publication was specifically attributed to CP.
JP 60149529 relates to the production of differentiation-inducing factors, as a result of administration of CP to mammals. In addition, medicines for leukemia in which the active ingredients are the differentiation-inducing factors produced after treating mammals with CP are described. As indicated in this publication, the differentiation of leukemic cells obtained by the differentiation-inducing factors is induced via a CP stimulus. Serum obtained from rabbits which repeatedly received CP was capable of inducing differentiation of M1 cells into macrophages. However, CP by itself was incapable of inducing the differentiation. There is no indication as to the identity or nature of the substance obtained by CP stimulus, which causes the induction of differentiation.
The use of CP has been described also for the preparation of other pharmaceutical compositions. For example, GB 1,304,697 describes pharmaceutical compositions comprising CP for use, in particular, against inflammation.
Further, clinical trials have shown that CP may be helpful in therapy of aplastic anemia [Shimizu, M., Transfusion 19(6):742-8 (1979); Arimori, S., Jap. J. Clin. Exper. Med. 43:1897 (1966)].
Contrary to the earlier reports, the present invention reveals that the activities previously ascribed to CP, should be attributed to a small molecular weight composition of matter (SDF), which in serum is preferentially associated with CP (SDF-CP complex). Regardless of the nature of association between SDF and CP, it is clear that CP derived from adult serum is a rich source of the factor. Other sources for SDF are urine and fetal serum. Due to its large molecular weight, intact CP cannot be present in the urine. Thus, it is assumed that the factor present in urine is not associated with the CP molecule, or at least not with the intact molecule.
As will be shown hereafter, SDF, as well as its complex with CP, which are the subject of the present invention, may have many therapeutical uses.
The present invention relates to a biologically active serum-derived composition of matter (SDF), having a low molecular weight, being electrically charged at acidic pH and having absorption at 280 nm. The molecular weight of SDF, as determined by electron spray is 316.
In a second aspect, the invention relates to a method for the isolation from plasma and purification of a low molecular weight composition of matter, comprising the steps of (a) transferring plasma through an affinity column to obtain an electrophoretically homogeneous fraction being the SDF-CP complex, which may be optionally concentrated by transferring through an anion exchange column; (b) isolating the SDF from the SDF-CP complex by either (i) transferring the fraction obtained in step (a) through RP-HPLC xe2x80x9cResourcexe2x80x9d(trademark) column, elution buffer A consisting of 0.05-0.1% trifluoroacetic acid (TFA) in water (pH 2.5), elution buffer B consisting of acetonitrile, the fractions eluted at acetonitrile concentration of 0-2% and 13-17% being collected and combined, or (ii) extracting the fraction obtained in step (a) with an acidified solvent, wherein the active fraction is recovered from the organic phase; (c) purifying the fraction obtained in (b) by RP-HPLC chromatography separation using a C-18 column, wherein in a first optional separation step elution buffer A, consisting of 0.1% TFA in water (pH 2.5), and elution buffer B, consisting of 0.1% TFA in acetonitrile, are employed, the fraction eluted at acetonitrile concentration of 0-2% being collected, and in the subsequent separation step, elution buffer A, consisting of 0.1% triethylamine in water and adjusted to pH 7.0, and elution buffer B, consisting of acetonitrile, are employed, the fraction eluted at acetonitrile concentration of 9-11% being collected.
Also within the scope of the invention is a biologically active complex comprising ceruloplasmin and said biologically active composition of matter (SDF).
In a second aspect, the invention relates to pharmaceutical composition comprising as active ingredient the SDF of the invention or its complex with CP, and optionally further comprising pharmaceutically acceptable additives.
Such pharmaceutical compositions may be used for treatment of patients with aplastic marrow, for inducing or maintaining remission of tumors, for expanding hematopoietic normal stem and progenitor cells for bone marrow transplants or for inhibiting enhanced angiogenesis.